JP2002233363A - Method for stabilizing ascorbate oxidase and reagent for assaying biocomponent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a long-term stable reagent for assaying biological fluid components the without interference of ascorbic acid. SOLUTION: This method for stabilizing ascorbate oxidase comprising formulating a solution containing the ascorbate oxidase with one or more kinds of carboxylic acids (salts) capable of stabilizing the ascorbate oxidase. The reagent for assaying the biological fluid components comprises the ascorbate oxidase and one or more kinds of the carboxylic acids (salts) capable of stabilizing the ascorbate oxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for stabilizing ascorbate oxidase and a reagent for measuring a biological component.

[0002]

2. Description of the Related Art Ascorbic acid is often added to foods for the purpose of preventing the oxidation of foods due to its strong reducing power. In addition, it is known that the ingestion thereof increases the concentration of serum, urine and the like in a living body. Therefore, it is known that in the field of analysis of foods to which ascorbic acid has been added or in clinical tests for analyzing components in biological samples such as serum and urine, measurement can be hindered by ascorbic acid in measurement samples. Various methods for avoiding the problem have been reported. Among them, a method of containing ascorbate oxidase in a measurement reagent and oxidizing ascorbic acid derived from a sample to avoid the influence is often used.

[0003]

Since ascorbate oxidase is easily deactivated in a solution, there is a disadvantage that the resolution of ascorbic acid is reduced during storage of the reagent.
As a means to prevent a decrease in resolution of ascorbic acid,
Various stabilizers have been added to ascorbate oxidase. Examples of stabilizers include boric acid,
Sucrose and the like, and the stability of ascorbate oxidase in solution is improved by these methods. However, it has not been possible to completely avoid interference with the measurement of ascorbic acid, and it has been difficult to maintain the ability to avoid the interference of ascorbic acid for a long time in a liquid state.

[0004]

Means for Solving the Problems In view of the above-mentioned situation, the present inventors have intensively studied a method capable of avoiding interference with measured values of ascorbic acid for a long period of time.
The coexistence of ascorbate oxidase and carboxylic acid and / or a salt thereof (hereinafter abbreviated as carboxylic acid (salt)) stabilizes ascorbate oxidase, and has solved the above-mentioned problem.

[0005] That is, the present invention relates to a method for stabilizing ascorbate oxidase, which comprises mixing one or more carboxylic acids (salts) with a solution containing ascorbate oxidase. The present invention also relates to a reagent for measuring a biological component containing ascorbate oxidase and one or more carboxylic acids (salts).

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The ascorbate oxidase used in the present invention is not particularly limited.
Examples thereof include those obtained from cucumber plants such as pumpkin and cucumber, and those derived from microorganisms. In addition, it includes those produced by genetic recombination technology and those chemically modified.

The carboxylic acids (salts) used in the present invention include hydroxypolycarboxylic acids (salts) such as L-tartaric acid and sodium L-tartrate, potassium L-tartrate and sodium potassium L-tartrate. , Citric acid, sodium citrate and the like. Examples of the dicarboxylic acid include dicarboxylic acids such as succinic acid, 3,3-dimethylglutaric acid, adipic acid and α-ketoglutaric acid, and salts thereof.

Examples of the monocarboxylic acid include monocarboxylic acids such as pyruvate, sodium pyruvate, lithium pyruvate, and propionic acid, and salts thereof.
These carboxylic acids (salts) can be used singly or in combination of two or more, but pyruvic acid (salt) such as pyruvic acid, sodium pyruvate and lithium pyruvate is preferred.

The reagent for measuring a biological component of the present invention comprises at least ascorbate oxidase and carboxylic acid (salt).
As long as it is a solution containing the compounds, the composition of the reagent may be one solution, two or more solutions, or a lyophilized form. If the concentration of the carboxylic acid (salt) in this reagent is 0.1% by weight or more, the effect of stabilizing ascorbate oxidase can be obtained, but preferably 0.1 to 10% by weight, more preferably 0.5%. ~ 5.0% by weight.

[0010] The reagent for measuring a biological component which can be a target in the present invention is not particularly limited,
There is a system using a known oxidase. Specific examples include a direct or total bilirubin measurement reagent using bilirubin oxidase, a glucose measurement reagent using glucose oxidase and peroxidase (hereinafter abbreviated as POD), a free cholesterol measurement reagent using cholesterol oxidase and POD, Glycerol kinase, glycerol-3-phosphate oxidase and POD
, A phospholipase D, choline oxidase and POD-based phospholipid measurement reagent, creatine amidinohydrase, sarcosine oxidase and POD-based creatinine measurement reagent, uricase and P
It is a uric acid measurement reagent using OD, and the like.

Hereinafter, the reagent for direct bilirubin measurement will be specifically described. The buffer used for this reagent is pH
6.5 or less is suitable, and preferably pH 4.7 to 6.5.
Anything that has a buffer capacity between the two may be used. For example, phthalic acid-sodium hydroxide buffer, malic acid-
Sodium hydroxide buffer, citric acid-sodium citrate buffer and the like. Buffer concentration is 10-500
mM is suitable, preferably 30-200 mM.

The bilirubin oxidase includes, for example, an enzyme obtained from a strain belonging to the genus Milosecium or the genus Trachiderma, and the amount of the enzyme used is 0.01 to 0.01%.
200 units / ml is suitable, preferably 0.1 to 20 units / m
l. Ascorbate oxidase used in the present invention is not particularly limited, for example, pumpkin,
Examples include those obtained from cucumber and other cucumber plants and those derived from microorganisms. In addition, it includes those produced by genetic recombination technology and those chemically modified. Its concentration is between 0.1 and 100
Units / ml are appropriate.

The carboxylic acids (salts) used in the measuring reagent of the present invention include hydroxypolycarboxylic acids (salts) such as L-tartaric acid and sodium L-tartrate, potassium L-tartrate and sodium potassium L-tartrate. Citric acid (salt) such as tartaric acid (salt), citric acid, and sodium citrate; Dicarboxylic acids include succinic acid, 3,3-dimethylglutaric acid, adipic acid, α-
Examples include dicarboxylic acids such as ketoglutaric acid and salts thereof.

Examples of the monocarboxylic acids include monocarboxylic acids such as pyruvic acid, sodium pyruvate, lithium pyruvate and propionic acid, and salts thereof.
These carboxylic acids (salts) can be used alone or in combination of two or more, but pyruvic acid (salt) such as pyruvic acid, sodium pyruvate and lithium pyruvate is preferred. If the concentration of the carboxylic acid (salt) in this reagent is 0.1% by weight or more, the effect of stabilizing ascorbate oxidase can be obtained, but preferably 0.1 to 10% by weight, more preferably 0.5%. ~ 5.0% by weight.

Other components include fluoride salts of alkali metals and the like having a thiol group, which are necessary for the separation of direct bilirubin, and p-toluene for eliminating measurement error factors originating from the sample and for accurate measurement. Sulfonic acid,
Aromatic carboxylic acids such as benzoic acid, surfactants, amino acids such as alanine and serine, sugars such as mannitol, polyols such as polyethylene glycol, salts such as NaCl, proteins such as albumin, and preservatives can be added. it can.

In the present invention, a reagent for direct bilirubin measurement can be obtained by appropriately selecting the above-mentioned components and mixing them by a conventional method. In a preferred embodiment, the first solution is 10-200 mM phthalate buffer adjusted to pH 4.7-6.5, Triton X-100 0.005.
~ 0.5 wt%, alanine 5-100 mM, p-toluenesulfonic acid 1-100 mM, human serum albumin 0.001-
1% by weight, containing 0.05 to 200 mM sodium fluoride and 0.02 to 10 mM N-acetylcysteine;
The solution contains 0.1 to 100 units / ml of ascorbate oxidase, 0.1 to 10% by weight of carboxylic acid (salt), 0.05 to 100 units / ml of bilirubin oxidase, and pH 4.0.
10-200 mM phthalate buffer prepared to 7-6.5
including.

A specific method for measuring direct bilirubin using the thus obtained reagent of the present invention (two-part system) will be described below. First, the first liquid for direct bilirubin measurement described above
(0.6 ml), an appropriate amount of a sample (serum, etc.) containing various bilirubins is added, and after preheating in a spectrophotometer cell chamber, 460 nm
The absorbance at is measured. The above-mentioned second solution for direct bilirubin measurement (0.15 ml) was added thereto, and the mixture was reacted for 1 to 10 minutes to oxidize only direct bilirubin.
The absorbance at 60 nm is measured, and the absorbance change (A) is determined from this value and the liquid amount correction value of the absorbance previously measured.
Next, a standard substance containing a known concentration of direct bilirubin is measured in the same manner, and the amount of change in absorbance (B) is determined. The direct bilirubin content in the sample can be determined from the following equation from the change in the absorbance determined by the action of the sample and the change in the absorbance determined by the action of the standard substance. Direct bilirubin concentration in the sample (mg / dl) = (A / B) x direct bilirubin concentration in the standard substance (mg / dl)
0.1 ml is preferred. The measurement wavelength is not limited to 460 nm, and any wavelength between 400 and 480 nm can be selected. In addition, the liquid amounts of the first liquid, the second liquid, and the sample can be appropriately changed.

Next, the reagent for measuring total bilirubin will be described. The buffer used for this reagent is suitably pH 7 to 11, and preferably any buffer having a buffering capacity between pH 7.5 to 10. For example, Tris-HCl buffer, phosphate buffer, borane Buffers and the like can be mentioned. The concentration of the buffer is suitably 10 to 500 mM, preferably 30 to 2 mM.
00 mM.

The bilirubin oxidase includes, for example, an enzyme obtained from a strain belonging to the genus Myrocesium or the genus Trachiderma.
200 units / ml is suitable, preferably 0.1 to 20 units / m
l.

Other components include compounds having a thiol group, aromatic carboxylic acids such as p-toluenesulfonic acid and benzoic acid, surfactants, amino acids such as alanine and serine, sugars such as mannitol, and polyols such as polyethylene glycol. , Salts such as NaCl, proteins such as albumin and the like can be added as necessary.

In the present invention, a reagent for measuring total bilirubin can be obtained by appropriately selecting the above components and mixing them by a conventional method.

Next, measuring reagents for measuring biological components other than bilirubin, that is, glucose measuring reagents using glucose oxidase and POD, free cholesterol measuring reagents using cholesterol oxidase and POD, glycerol kinase and glycerol-3 A reagent for measuring neutral fats using phosphate oxidase and POD, a reagent for measuring phospholipids using phospholipase D, choline oxidase and POD, a reagent for measuring creatinine using creatine amidinohydrase, sarcosine oxidase and POD, uricase and POD Reagents based on the principle of performing a color reaction in combination with oxidase and POD, such as a uric acid measurement reagent using oxidase, include oxidase, POD, chromogen, coupler and ascorbate oxidase, and carboxylic acid (salt).

As the POD used here, horseradish-derived POD can be used. The coloration of hydrogen peroxide generated by each oxidase requires a chromogen and a coupler, and the chromogen is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyl. Aniline (TOOS), N-acetylcysteine, phenol,
2-chlorophenol, 4-chlorophenol, 2,4
Phenol derivatives such as dichlorophenol, or aniline, N, N-dimethylaniline, N, N-diethylaniline, N, N-diethyl-m-toluidine, N, N
-Diethyl-m-anisidine, N-ethyl-N- (3-
Methylphenyl) -N'-acetylethylenediamine,
N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine, N-ethyl-N-sulfopropyl Aniline derivatives such as -m-toluidine and N-ethyl-N-sulfopropyl-m-anisidine; Also, as a coupler,
4-aminoantipyrine, 3-methyl-2-benzothiazolinone hydrazone, and the like. Oxidase,
The amounts of peroxidase, chromogen, and coupler are not particularly limited as long as they do not interfere with the measurement.

[0024]

The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In the examples, abbreviations indicate the following. TOOS: N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethylaniline, ATP: adenosine triphosphate, BES: N, N'-bis (2-hydroxyethyl) -2- Aminoethanesulfonic acid, MES: 2- (morpholino) ethanesulfonic acid, TES: N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, TAPS: N-tris (hydroxymethyl) methyl-3-
Aminopropanesulfonic acid, EDTA: disodium ethylenediaminetetraacetate,

Example 1 Free Cholesterol in Serum
The following reagents A to C were prepared. Reagent A: (First reagent) Peroxidase 10 U / ml, TOO
S 2.0 mM, MES 100 mM, pH 6.3 (Second reagent) Cholesterol oxidase 1.5 U /
ml, 4-aminoantipyrine 0.6 mM, MES 1
00 mM, pH 6.3 Reagent B: (first reagent) peroxidase 10 u / ml, TOO
S 2.0 mM, MES 100 mM, pH 6.3, ascorbate oxidase 2 U / ml (from pumpkin) (Second reagent) cholesterol oxidase 1.5 U / m
1, 4-aminoantipyrine 0.6 mM, MES 10
0 mM, pH 6.3 Reagent C: (First reagent) Peroxidase 10 U / ml, TOO
S 2.0 mM, MES 100 mM, pH 6.3, ascorbate oxidase 2 U / ml, sodium pyruvate 1.0% (second reagent) cholesterol oxidase 1.5 U /
ml, 4-aminoantipyrine 0.6 mM, MES 1
00 mM, pH 6.3 Further, reagents A to C which were allowed to stand at 10 ° C. for one year were designated as reagents A ′ to C ′, respectively.

The following samples 1 to 6 were prepared as samples. (Sample 1) Human pool serum: distilled water = 9: 1, (Sample 2) Human pool serum: 200 mg / dl ascorbic acid aqueous solution = 9: 1, (Sample 3) Human pool serum: 400 mg / dl ascorbic acid aqueous solution = 9 : 1, (Sample 4) human pool serum: 600 mg / dl ascorbic acid aqueous solution = 9: 1, (Sample 5) human pool serum: 800 mg / dl ascorbic acid aqueous solution = 9: 1, (Sample 6) human pool serum: 1000 mg / dl Ascorbic acid aqueous solution = 9: 1.

Each of the reagents A to C ′ (A, A ′, B, B ′, C ′) was added to 4 μl of each sample corresponding to each of the samples 1 to 6.
And C '; the same applies hereinafter).
After addition of μl and heating at 37 ° C. for 5 minutes, 100 μl of the second reagents A to C ′ were added, and the mixture was heated at 37 ° C. for 5 minutes and the absorbance at 546 nm was measured using purified water as a control. Based on the obtained absorbance, the free cholesterol concentration was determined from a calibration curve previously prepared using a standard solution. Table 1 shows the results. In the table, numbers indicate units of mg / dl.

[0028]

[Table 1]

As is clear from Table 1, free cholesterol cannot be measured accurately with reagents A and A 'due to the influence of ascorbic acid. In the case of reagent B containing only ascorbate oxidase, interference with ascorbic acid can be avoided when the storage treatment is not performed at 10 ° C. for one year, but in the case of reagent B ′ stored for a long time, interference with high concentration of ascorbic acid is avoided. Ability was lost. However, the effect of the reagent C of the present invention can be avoided even in a sample having a high ascorbic acid concentration before and after storage.

Example 2: Measurement of uric acid in serum The following reagents DF were prepared. Reagent D: (first reagent) peroxidase 10 U / ml, TOO
S 2.0 mM, BES 100 mM, pH 7.0, (second reagent) uricase 1 U / ml, 4-aminoantipyrine 0.6 mM, BES 100 mM, pH 7.0, reagent E: (first reagent) peroxidase 10 U / ml, TOO
S 2.0 mM, BES 100 mM, pH 7.0, ascorbate oxidase 2 U / ml (from Acremonium sp.) (Second reagent) Uricase 1 U / ml, 4-aminoantipyrine 0.6 mM, BES 100 mM, pH 7.0 Reagent F: ( 1st reagent) Peroxidase 10U / ml, TOO
S 2.0 mM, BES 100 mM, pH 7.0, ascorbate oxidase 2 U / ml (from Acremonium sp.), Sodium pyruvate 0.2% (second reagent) uricase 1 U / ml, 4-aminoantipyrine, 0.6 mM BES 100 mM, pH 7.0 In the same manner as in Example 1, each of the reagents D ′ to F ′ was prepared by leaving each reagent at 10 ° C. for one year. The same samples 1 to 6 as in Example 1 were prepared as samples.

In 6 μl of each sample, the first reagent of each of the reagents DF ′ was added to 200 μl corresponding to each of the samples 1 to 6.
After heating at 37 ° C. for 5 minutes, 100 μl of the second reagent of each of the reagents DF ′ was added, and after heating at 37 ° C. for 5 minutes,
Absorbance at 546 nm was measured using purified water as a control. Based on the obtained absorbance, the uric acid concentration was determined from a calibration curve previously prepared using a standard solution. Table 2 shows the results. In the table, numbers indicate units of mg / dl.

[0032]

[Table 2]

As is evident from Table 2, even in a sample having a high ascorbic acid concentration, the reagent F of the present invention can avoid interference of interfering substances for a long period of time.

Example 3 Measurement of Neutral Fat The following reagents G to I were prepared. Reagent G: (first reagent) glycerol kinase 1 U / ml, glycerol-3-phosphate oxidase 5 U / ml, peroxidase 10 U / ml, TOOS 2 mM, ATP
3 mM, TES buffer 50 mM, pH 7.0, (second reagent) lipoprotein lipase 3 U / ml, 4
-Aminoantipyrine 0.6 mM, TES buffer 50
mM, pH 7.0, Reagent H: (first reagent) glycerol kinase 1 U / ml, glycerol-3-phosphate oxidase 5 U / ml, peroxidase 10 U / ml, TOOS 2 mM, ATP
3 mM, TES buffer 50 mM, pH 7.0, ascorbate oxidase 2 U / ml (from Acremonium sp.) (Second reagent) lipoprotein lipase 3 U / ml, 4
-Aminoantipyrine 0.6 mM, TES buffer 50
mM, pH 7.0 Reagent I: (first reagent) glycerol kinase 1 U / ml, glycerol-3-phosphate oxidase 5 U / ml, peroxidase 10 U / ml, TOOS 2 mM, ATP
3 mM, TES buffer 50 mM, pH 7.0, ascorbate oxidase 2 U / ml (from Acremonium sp.), Sodium pyruvate 1.0% (second reagent) lipoprotein lipase 3 U / ml, 4
-Aminoantipyrine 0.6 mM, TES buffer 50
mM, pH 7.0 As in Example 1, reagents G′〜 stored at 10 ° C. for one year
I 'was prepared. In addition, the same samples 1 to 6 as in Example 1 were prepared as samples.

To 3 μl of each sample, 250 μl of the first reagent of each of the reagents GI ′ was added corresponding to each of the samples 1 to 6.
After heating at 37 ° C. for 5 minutes, the second reagent GI ′ was added.
After adding 125 μl of the reagent and heating at 37 ° C. for 5 minutes, the absorbance at 546 nm was measured using purified water as a control. Based on the obtained absorbance, the neutral fat concentration was determined from a calibration curve previously prepared using a standard solution. Table 3 shows the results. The numerical values in the table indicate units of mg / dl.

[0036]

[Table 3]

As is clear from Table 3, even in a sample having a high ascorbic acid concentration, the reagent I of the present invention can avoid the influence of the interfering substance for a long period of time.

Example 4: Measurement of serum phospholipids The following reagents J to L were prepared. Reagent J: (first reagent) phospholipase D 0.5 U / ml, peroxidase 10 U / ml, TOOS 2 mM, Tris buffer 50 mM (second reagent) choline oxidase 10 U / ml, 4-
Aminoantipyrine 0.6 mM, Tris buffer 50 m
M, pH 7.7

Reagent K: (first reagent) phospholipase D 0.5 U / ml, peroxidase 10 U / ml, TOOS 2 mM, Tris buffer 50 mM, pH 7.7, ascorbate oxidase 2 U / ml (from Acremonium sp.) (Second reagent) choline oxidase 10 U / ml, 4-
Aminoantipyrine 0.6 mM, Tris buffer 50 m
M, pH 7.7

Reagent L: (First reagent) Phospholipase D 0.5 U / ml, peroxidase 10 U / ml, TOOS 2 mM, Tris buffer 50 mM, pH 7.7, ascorbate oxidase 2 U / ml, sodium pyruvate 1. 0% (second reagent) choline oxidase 10 U / ml, 4-
Aminoantipyrine 0.6 mM, Tris buffer, pH
7.7 Reagents J '~ stored at 10 ° C for 1 year as in Example 1
L 'was prepared. The same samples 1 to 6 as in Example 1 were prepared as 50 mM samples.

To 3 μl of each sample, add 250 μl of the first reagent of reagents J to L ′ corresponding to each of samples 1 to 6.
After heating at 37 ° C. for 5 minutes, the second
After adding 125 μl of the reagent and heating at 37 ° C. for 5 minutes, the absorbance at 546 nm was measured using purified water as a control. Based on the obtained absorbance, the phospholipid concentration was determined from a calibration curve previously prepared using a standard solution. Table 4 shows the results. The numerical values in the table indicate units of mg / dl.

[0042]

[Table 4]

As is clear from Table 4, even in a sample having a high ascorbic acid concentration, the reagent L of the present invention can avoid the influence of the interfering substance for a long period of time.

Example 5: Measurement of serum creatinine The following reagents MO were prepared. Reagent M: (first reagent) creatine amidinohydrolase 100
U / ml, sarcosine oxidase 10 U / ml, peroxidase 10 U / ml, TOOS 2 mM, TA
PS buffer 50 mM, pH 8.0 (second reagent) creatinine amidohydrolase 300
U / ml, 4-aminoantipyrine 0.6 mM, TAP
S buffer 50 mM, pH 8.0

Reagent N: (first reagent) creatine amidinohydrolase 100
U / ml, sarcosine oxidase 10 U / ml, peroxidase 10 U / ml, TOOS 2 mM, TA
PS buffer 50 mM, pH 8.0, ascorbate oxidase 2 U / ml (from Acremonium sp.) (Second reagent) creatinine amidohydrolase 300
U / ml, 4-aminoantipyrine 0.6 mM, TA
PS buffer 50 mM, pH 8.0

Reagent O: (first reagent) creatine amidinohydrolase 100
U / ml, sarcosine oxidase 10 U / ml, peroxidase 10 U / ml, TOOS 2 mM, TA
PS buffer 50 mM, pH 8.0, ascorbate oxidase 2 U / ml (from Acremonium sp.), Sodium pyruvate 1.0% (second reagent) creatinine amidohydrolase 300
U / ml, 4-aminoantipyrine 0.6 mM TAP
S-buffer, pH 8.0 Reagents stored at 10 ° C. for 1 year as in Example 1
O 'was prepared. The same samples 1 to 6 as in Example 1 were prepared as 50 mM samples.

To 6 μl of each sample, 300 μl of the first reagent of each of the reagents M to O ′ was added corresponding to each of the samples 1 to 6.
After heating at 37 ° C. for 5 minutes, the second
After adding 100 μl of the reagent and heating at 37 ° C. for 5 minutes, the absorbance at 546 nm was measured using purified water as a control. Based on the obtained absorbance, the creatinine concentration was determined from a calibration curve previously prepared using a standard solution. Table 5 shows the results.
In the table, numerical values indicate units of mg / dl.

[0048]

[Table 5]

As is evident from Table 5, the reagent O of the present invention can avoid the effects of interfering substances over a long period of time even in a sample having a high ascorbic acid concentration.

Example 6: Measurement of serum bilirubin directly The following reagents PT were prepared. Reagent P: (first reagent) potassium hydrogen phthalate buffer 120m
M, pH 5.5, N-acetylcysteine 2.5 mM E
DTA 0.1 mM, p-toluenesulfonic acid 50 mM,
NaF 2.5 mM (second reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, bilirubin oxidase 0.1 U / m
L, ascorbate oxidase 4U / mL

Reagent Q: (first reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, N-acetylcysteine 2.5 mM E
DTA 0.1 mM, p-toluenesulfonic acid 50 mM,
NaF 2.5 mM (second reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, bilirubin oxidase 0.1 U / m
L, ascorbate oxidase 4U / mL (Acremon
ium sp.), sodium pyruvate 0.5% Reagent R: (first reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, N-acetylcysteine 2.5 mM E
DTA 0.1 mM, p-toluenesulfonic acid 50 mM,
NaF 2.5 mM (second reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, bilirubin oxidase 0.1 U / m
L, ascorbate oxidase 4U / mL (Acremon
ium sp.), sodium pyruvate 1.0%

Reagent S: (first reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, N-acetylcysteine 2.5 mM E
DTA 0.1 mM, p-toluenesulfonic acid 50 mM,
NaF 2.5 mM (second reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, bilirubin oxidase 0.1 U / m
L, ascorbate oxidase 4U / mL (Acremon
ium sp.), sodium pyruvate 2.0% Reagent T: (first reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, N-acetylcysteine 2.5 mM E
DTA 0.1 mM, p-toluenesulfonic acid 50 mM,
NaF 2.5 mM (second reagent) potassium hydrogen phthalate buffer 120 m
M, pH 5.5, bilirubin oxidase 0.1 U / m
L, ascorbate oxidase 4U / mL (Acremon
ium sp.), sodium pyruvate 5.0% In the same manner as in Example 1, reagents P ′ to T ′ which were allowed to stand at 25 ° C. for one month were prepared. The same samples 1 to 1 as the samples 1
6 were prepared.

The first reagent of each of the reagents P to T ′ is added to 14 μl of the sample for 280 corresponding to each of the samples 1 to 6.
After addition of μl and heating at 37 ° C. for 5 minutes, 70 μl of each of the reagents P to T ′ was added and heated at 37 ° C. for 5 minutes, and the absorbance at 450 nm was measured using purified water as a control. Based on the obtained absorbance, the bilirubin concentration was determined from a calibration curve previously prepared using a standard solution. Table 7 shows the results. In the table, numbers indicate units of mg / dl.

[0054]

[Table 6]

As is clear from Table 6, the reagents Q to T of the present invention can measure serum bilirubin directly over a long period of time even in a sample having a high ascorbic acid concentration while avoiding the effects thereof.

[0056]

According to the present invention, ascorbate oxidase can be stabilized in a liquid state for a long time by a very simple and economical method of adding a carboxylic acid, and the ascorbic acid contained in a sample can be stabilized. The components in the target biological fluid can be accurately quantified without being affected.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Mitsuo Watanabe 23 Unit Uji Kozakura, Uji City, Kyoto Prefecture Unitika Central Research Laboratory F Term (Reference) 4B050 HH02 KK06 LL03 4B063 QA01 QR03 QR41 QR53 QS24

Claims (8)

[Claims] 1. A method for stabilizing ascorbate oxidase, comprising mixing one or more carboxylic acids (salts). 2. The method for stabilizing ascorbate oxidase according to claim 1, wherein the carboxylic acid (salt) is hydroxypolycarboxylic acid, dicarboxylic acid, monocarboxylic acid or a salt thereof. 3. The method for stabilizing ascorbate oxidase according to claim 1, wherein the carboxylic acid (salt) is succinic acid, pyruvic acid or a salt thereof. 4. The method for stabilizing ascorbate oxidase according to claim 1, wherein the concentration of carboxylic acids (salts) in the solution containing ascorbate oxidase is 0.1% by weight or more. 5. A reagent for measuring a biological component in a body fluid, comprising ascorbate oxidase and one or more carboxylic acids (salts). 6. The reagent according to claim 5, wherein the carboxylic acid (salt) is hydroxypolycarboxylic acid, dicarboxylic acid, monocarboxylic acid or a salt thereof. 7. The reagent for measuring a biological component according to claim 5, wherein the carboxylic acid (salt) is succinic acid, pyruvic acid or a salt thereof. 8. The reagent for measuring a biological component according to claim 5, wherein the concentration of the carboxylic acid (salt) in the reagent is 0.1% by weight or more.